It is now obligated to do reproductive and developmental toxicity studies in studies relating to safety of a drug on the occasion of an application of manufacturing approval for the drug. The reproductive and developmental toxicity studies mean that animal experiments are conducted to obtain information on whether the application of a drug to living bodies could possibly induce some adverse effect in the course of reproduction and development. The experimental results can be extrapolated to humans and utilized in the evaluation of safety (risk) of the drug to reproduction and development in humans. A guideline for reproductive and developmental toxicity studies promulgated in 1997 was revised and enacted in conformity with the ICH guideline (International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use; at the international conference for harmonization of regulation of pharmaceuticals in three areas involving Japan, USA and EU) which was advised on 1994. In this guideline, it is described that histopathological examinations of reproductive organs and a sperm analysis in repeated-dose toxicity studies should be made relative to evaluation of male fertility in studies relating to fertility and early embryonic development to implantation.
To perform the histopathological examination for male reproductive organs, however, skillful technique is required, and there is sometimes a case given an effect to spermatozoa which could not be detected by means of the histopathological examination. In order to correctly evaluate fertility and precisely examine an effect of a drug on spermatozoa, it is necessary to perform the fertility examination of spermatozoa themselves as well as the histopathological examination.
In the current reproductive and developmental toxicity studies, no examination of concentration, motility, morphology of spermatozoa and the like that have been performed in human sperm analysis. Accordingly, an investigation for establishing a method for evaluation of spermatozoa motility is now being continued. This means that a method for evaluation of fertility has not yet been established.
As for examinations of fertility of human spermatozoa, a hamster test, conserved zona pellucida passage test, and triple-stain test and the like have been developed. The hamster test is a method for evaluating fertility, in which the properties of spermatozoa are utilized, that is, only spermatozoa which have the capacitation and in which the acrosome reaction have been completed, can enter the zona-free hamster eggs. The conserved zona pellucida passage test is a method in which the properties of spermatozoa are utilized, that is, only spermatozoa which have the capacitation, in which the acrosome reaction have been completed and in which the motility is retained sufficiently, can pass through the conserved pellucid zone.
The triple-stain method is a method for distinguishing dead spermatozoa, live spermatozoa and spermatozoa of intact acrosome by means of staining them with a combination of three types of staining methods.
When the above-mentioned test that, so far, is known for human spermatozoa is applied to the reproductive and developmental toxicity studies, it is necessary to collect eggs to eliminate the zona pellucida, or to permits maturation of the collected immature eggs in vitro and preserve them in a particular salt solution at a high concentration to form the zona pellucida. These procedures, however, require skillful technique and troublesome operation as well as expensive equipment. When the influence of chemical materials such as drugs or environmental hormones on fertility of spermatozoa is examined, in the current reproductive and developmental toxicity studies the decision whether or not to be fertilized on an experimental animal is not determined from their appearance as long as the fetus has grown up to some degree after mating. This necessitates time and cost. Even though many points such as the number of spermatozoon, mobility and morphology, which appear to have some influence on fertilization, are examined, it is impossible to determine the exact fertility so long as the presence of acrosome reaction as an essential factor for fertilization cannot be confirmed.